Abstract

The major goal of the project was the investigation of proteins that regulate dynamic rearrangements of the actin cytoskeleton in Dictyostelium discoideum amoebae. Among the proteins studied in detail were (i) D. discoideum vasodilator stimulated phosphoprotein DdVASP and a new profilin isoform as putative regulators of filopodia formation, and (ii) the Ste20-like kinases Krs1 and Severin-Kinase as members of signalling cascades towards the actin cytoskeleton.
Filopodia are bundles of actin filaments projecting from the cell surface. They are found on a variety of cell types and are needed among others, for cell adhesion, sensory and exploratory functions. Filopodia are frequently found associated with sheet-like arrays of actin filaments called lamellipodia and membrane ruffles. The function of the VASP homolog from D. discoideum in filopodia formation was studied using molecular, biochemical and cell biological approaches. The protein sequence of DdVASP shares a significant homology to the members from other species. The protein harbours two Ena/VASP homology domains EVH1 and EVH2 separated by a polyproline stretch. The EVH2 domain is characterised by a G-actin binding site (GAB), an F-actin binding site (FAB) and a tetramerisation domain. As a tetramer the DdVASP protein can nucleate actin polymerization and bundle actin filaments. The in vitro nucleating activity of DdVASP is salt dependent and its nucleating activity is completely abolished at 100 mM salt. However, the F-actin bundling activity as determined by the low speed sedimentation assay was not disturbed.
The ability of DdVASP to influence the binding of capping protein to the growing ends of the actin filaments was tested through elongation of capped F-actin seeds and by depolymerization of capped filaments upon dilution below the critical concentration of the barbed ends. Results from both sets of experiments showed that DdVASP cannot remove capping protein from the barbed ends. The D. discoideum formin dDia2, which was previously reported to be essential for filopodia formation could elongate the capped F-actin seeds. In vitro biochemical data led to the conclusion that the bundling activity of DdVASP is the essential in vivo function to stabilise actin filaments in emerging filopodia. To test this hypothesis, a DdVASP null mutant was isolated. As expected the mutant failed to produce any filopodia. Expression of wild type DdVASP, but not DdVASPFAB, rescued the phenotype suggesting the importance of the bundling activity of DdVASP in filopodia formation. To confirm that the data obtained with DdVASP were not species specific, key biochemical functions of HsVASP were also tested. The results indicated that VASPs are functionally well conserved throughout evolution.
During this study, a third profilin isoform, profilin III, was further characterised. Specific interaction between profilin III and DdVASP was discovered. Profilin III shares a limited homology at the amino acid level with the other two and well studied profilins. Polyclonal antibodies that recognise only the profilin III isoform showed that in wild type cells profilin III represents less than 1% of all profilins. This suggests a distinct role for profilin III, because a low protein concentration argues against an actin sequestering function. Immunolocalisation studies showed profilin III in filopodia and enriched at their tips. Cells lacking the profilin III protein show defects in cell motility during chemotaxis.
The second part of the project dealt with the characterisation of two D. discoideum Germinal Centre Kinases (GCK). The catalytic domain of Krs1 was found to be highly homologous to the catalytic region of human MST1 and MST2 from the GCK-II subfamily. The regulatory region harbours the putative inhibitory domain (aa 330-379) and a possible multimerization (SARAH) domain (aa 412-458) described for GCKs in higher organisms. This SARAH region spans about 50 amino acid residues, is located at the extreme carboxyl terminus and most likely forms an  - helical coiled-coil motif. GFP-Krs1 overexpressing wild type cells showed an enrichment of the kinase in the cell cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild type cells. Severin kinase is encoded by the gene svkA, and phylogenetic analysis groups it into subfamily GCK-III, along with human MST3, MST4 and YSK/SOK-1. Immunoblot analysis with polyclonal antibodies showed an uniform expression level throughout development. Gene disruption of svkA resulted in cells that had problems to divide both in submerged or in shaking cultures. Though the motility and chemotaxis of these cells remain unaltered compared to the wild type cells, the movement of the multicellular slugs is disturbed. In addition, development was delayed and the mutant formed aberrant fruiting bodies.